The nicotinic acetylcholine receptor (AChR) is a multimeric, integral membrane glycoprotein that functions as a ligand gated channel at the neuromuscular junction. The biosynthesis, assembly, membrane insertion and localization of the AChR complex provides an attractive system for the study of the mechanisms underlying these important cellular functions. Three complementary approaches will be used to provide additional fundamental information concerning AChR biogenesis, regulation, structure and function. 1) We plan to extend our monoclonal antibody (mAb) studies to focus on the extracellular domains of mouse muscle AChR including the ligand binding site and to identify the characteristics of cross-reacting antigens observed in a variety of electrically excitable tissues, including Drosophila central nervous system (CNS), guinea pig ileum smooth muscle, rat sympathetic ganglia, and the rat pheochromocytoma PC12 cell line. 2) Somatic cell genetic techniques will be applied to the questions of AChR regulation and structure-function relationship. Fluorescence-activated cell sorting in combination with specific immunotoxins and replica-plating will be used to obtain muscle cell variants with altered regulatory, biosynthetic, or structural features involving the AChR. 3) The protein-blotting approach will be used to provide additional structural information on the AChR as well as on possible evolutionarily related structures such as the Alpha-bungarotoxin (BuTX) binding site in the CNS of lower vertebrates. We plan also to utilize this approach to identify the amino acid sequences making up the epitopes responsible for various cross-reactions and to thus map the immunologically identifiable sites with respect to other structural features of the AChR.
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